The synthesis and aromatase inhibitory profile of 6 alpha and 6 beta-propargyl androstenedione and estrenedione are described. The targeted compounds 1 and 2 were prepared by addition of the propargyl Gringard to the 5 alpha,6 alpha-epoxy bisketal 6 or the 5 alpha,6 alpha-epoxy diacetate 7 followed by dehydration of the 6 beta-propargyl 5 alpha-hydroxy diones 10 and 11 using thionyl chloride. Treatment of the 6 beta-propargyl analogs 1 and 2 with hydrochloric acid gave the corresponding 6 alpha-propargyl isomers 3 and 4. Inhibitory activity of the synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H]-4-androstenedione as substrate. Under initial velocity assay condition of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent KiS ranging from 10 to 66 nM, with the Km for androstenedione being 55 nM. 6 alpha-Propargylandrost-4-ene-3,17-dione and 6 alpha-propargylestr-4-ene-3,17-dione were found to be potent competitive inhibitors of aromatase (Ki 37 and 66 nM, respectively). On the other hand the 6 beta-propargylandrost-4-ene-3,17-dione (6 beta-PAD) and 6 beta-propargylestr-4-ene-3,17-dione (6 beta-PED) were found to bind to aromatase with an apparent Ki of 10 and 48 nM, respectively, as well as cause rapid time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no inactivation was observed in the absence of NADPH. Substrate protects the enzyme from inactivation, but beta-mercaptoethanol does not, suggesting that the 6 beta-propargyl analogs 6 beta-PAD and 6 beta-PED are mechanism-based inactivators of aromatase. Energy-minimization calculations and molecular modeling studies indicate three global minima for each of the 6 beta-propargyl analogs in which one of the conformers is proposed to be responsible for the inactivation of aromatase.